Atrial fibrillation (AF) is a very common arrhythmia contributing to morbidity and mortality of affected patients (Wolf et al., 1998). Medical treatment remains a mainstay of therapy (Nattel and Opie, 2006), although the use of antiarrhythmic drugs is limited by potentially deleterious side effects (Hohnloser and Singh, 1995). Porcine models have been used to test Kv1.5-targeting atrial-selective drugs that are designed to circumvent this proarrhythmic risk inherent to treatment with conventional antiarrhythmic drugs (Wirth et al., 2003). However, to date there has been no clear demonstration of Kv1.5-related currents in porcine atrium.

Potassium currents [in particular transient outward currents (Ito) and IKur] are among the targets of novel atrialselective drugs developed for treating AF (Knobloch et al., 2004). Kinetic differences of activation, inactivation and recovery from inactivation discriminate between various potassium currents and are related to properties of the underlying ion channel subunits. The properties of Ito have been characterized extensively in many species (Patel and Campbell, 2005). Fast-inactivating Ito (Ito,f) is carried by Kv4.3 subunits in dog and human, whereas Kv1.4 subunits contribute to Ito,f in rabbit atria (Wang et al., 1999; Patel and Campbell, 2005). Rabbit Ito is characterized by particularly slow recovery from inactivation, which is related to the participation of Kv1.4 subunits (Wang et al., 1999). In human atrium, IKur is carried by Kv1.5 subunits, a member of the delayed rectifier current family, and has been shown to inactivate slowly over a period of seconds (Feng et al., 1998; Nattel et al., 1999). At room temperature, IKur may show a noninactivating phenotype (Li et al., 2004) although recording conditions importantly modulate inactivation kinetics of this current (Snyders et al., 1993). Likewise, β-subunits may modify inactivation properties of Kv1.5 currents (Uebele et al., 1996).

Porcine atrial cellular electrophysiology has been studied to a limited extent and is poorly understood, although this species is commonly used in experimental studies (Janse et al., 1998). A recent investigation demonstrated the presence of a Ca2+-dependent chloride current (ICl,Ca) and IKur in porcine atria (Li et al., 2004). In preliminary studies, we noted a robust time-dependent current in porcine atrium that activates rapidly (like IKur) and inactivates more slowly than classic Ito and somewhat more rapidly than previously reported IKur. This study aimed to characterize this porcine outward current (which we abbreviate IK,PO) with respect to electrophysiological properties, expression of potential corresponding underlying subunit transcripts, pharmacological responses, and functional role. In particular, we were interested in studying the pharmacological profile of the current with respect to known selective blockers of potential underlying K+ channel subunits, with a view to determining whether IK,PO can potentially account for previous reports of anti-AF actions of Kv1.5 blockers in pig hearts.

Materials and Methods

Animal and Tissue Handling. Male castrated pigs of the German landrace (n = 62; 19 ± 0.5 kg) were anesthetized with intravenous application of 30 mg/kg pentobarbital. During deep anesthesia, hearts were excised via left thoracotomy, resulting in humane euthanasia. Hearts were immediately immersed in oxygenated Tyrode's solution. All procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication 85-13, revised 1996) and were performed by technicians specifically trained and experienced in animal care.

For isolation of single cardiomyocytes, the proximal circumflex coronary artery was cannulated and the atrial preparation was perfused with oxygenated Tyrode's solution on a Langendorff apparatus. The perfusion solution was then switched to Ca2+-free Tyrode's solution until all contraction ceased (∼10 min), and 100 U/ml collagenase (type II; Worthington Biochemicals, Freehold, NJ)-containing Ca2+-free Tyrode's solution was used for cell isolation as reported previously (Gogelein et al., 2004). After isolation, cells were stored in a high-[K+] storage solution at room temperature and studied within 12 h. Only healthy-looking cells with clear cross-striations and sharp edges were used for electrophysiological measurements. For real-time RT-PCR measurements, aliquots of isolated atrial cardiomyocytes were used, whereas the remainder of the cell isolation was used for electrophysiological experiments on the same days.

Results

Voltage and Time Dependence. One-second depolarizing pulses from a holding potential of –80 mV to potentials between –60 and +60 mV (0.1 Hz; Fig. 1A) elicited rapidly activating outward currents showing time-dependent inactivation. Based on this observation and further evidence detailed below, we termed this current IK,PO for porcine outward potassium current. IK,PO amplitude was quantified as the difference between peak and end-pulse steady-state current unless stated otherwise. Threshold to current activation was positive to –20 mV, and myocytes had a mean ± S.E.M. IK,PO-density of 11.6 ± 1.6 pA/pF upon depolarization to +60 mV (n = 20; Fig. 1B).

Inactivation voltage dependence was examined with 1000-ms prepulses followed by 750-ms test pulses to +60 mV (n = 10 cells; Fig. 1C). Current amplitudes were normalized to current at –100 mV and plotted against the voltage of the conditioning pulse. Activation voltage dependence was determined from the IK,PO-voltage relationship, corrected for driving force according to the equation aV = IV /(Imax(V – Erev)), where aV and IV are the activation variable and IK,PO amplitude at voltage V, Imax is IK,PO amplitude at +60 mV, and reversal potential (Erev) was –69.2 ± 2.3 mV (obtained from deactivating tail currents recorded at potentials between –100 and –60 mV after brief, ∼2- to 5-ms depolarizations to +60 mV). Erev was corrected for liquid junction potentials. Voltages for half-maximal activation and inactivation (Boltzman fits), and corresponding slope factors were 16.8 ± 3.8 mV (slope of 15.4 ± 1.7) and –28.2 ± 2.9 mV (slope of –6.1 ± 1.5).

Recovery from inactivation was assessed with a paired pulse protocol with depolarizations (P1 and P2) to +60 mV at increasing P1-P2 intervals (Fig. 1F) and a holding potential of –80 mV. Current during P2 was normalized to current during P1 and showed biexponential recovery with time constants of 1.54 ± 0.73 s (τf) and 7.91 ± 1.78 s (τs; n = 9; Fig. 1G).

To study frequency dependence, cells were repetitively depolarized from –80 to +60 mV (410-ms pulses) in Tris-Cl-containing, Na+-free external solution. Currents during the 10th pulse were normalized to current during the first pulse, showing a frequency-dependent decline (n = 15; Fig. 1H). Similar experiments were performed in the presence of extracellular NaCl with 50-ms prepulses to –50 mV to inactivate INa. These recordings showed slightly greater frequency dependence, but they were qualitatively comparable with those obtained with Tris-Cl (n = 7; Fig. 1H). In Tris-Cl, IK,PO elicited with the 10th pulse were 46 ± 3% (0.5 Hz) of P1, declining to 13 ± 3% (2 Hz), compared with 38 ± 3% (0.5 Hz) and 7 ± 1% (2 Hz) in NaCl-containing solution (P = 0.07 and 0.05, respectively). The effect of equimolar Na+ replacement by Tris and prepulse protocols to inactivate INa in Na+ - containing solution on peak to steady-state current was subtle. In nine cells, IK,PO at +60 mV without prepulses averaged 8.3 ± 1.4 and 9.5 ± 1.4 pA/pF (P = N.S.) in the presence and absence of extracellular Na+, respectively. In Na+-containing Tyrode's solution, currents recorded at +60 mV with 50-ms prepulses to –50 mV, averaged 8.4 ± 1.5 compared with 9.3 ± 1.4 pA/pF without prepulses (P = N.S.).

Effects of Cl– and K+ Substitution on IK,PO. To assess the potential charge carrier of IK,PO, we investigated the effects of Cl– and K+ substitution on the current under study. The reversal potential of the current averaged –69.2 ± 2.3 mV (n = 3 cells). After correction for liquid-junction potentials this value was ∼72 mV, compatible with a predominantly K+-carried conductance.

IK,PO was also recorded in individual cells before and after replacement of external chloride by equimolar cyclamate to assess potential contributions of a previously described Ca2+-dependent Cl– current (Li et al., 2004). This intervention did not alter IK,PO (Fig. 2, A and B). Mean ± S.E.M. IK,PO density was similar over a range of voltages, averaging 7.1 ± 1.8 pA/pF at +60 mV in NaCl and 7.0 ± 1.9 pA/pF with Nacyclamate (n = 5; P = N.S.; Fig. 2C). The effects of intracellular K+ replacement by Cs+ were then determined. Currents were recorded from 10 cells with regular internal solution and from nine other cells isolated from the same pigs on the same days with equimolar substitution of CsCl for internal KCl. Intracellular K+ removal abolished IK,PO: currents at +60 mV averaged 12.1 ± 0.9 pA/pF (KCl) versus 0.2 ± 0.05 pA/pF (CsCl; P < 0.001; Fig. 2F). Together, these results strongly suggest that IK,PO is primarily a K+ current.

Biophysical current characterization. A, IK,PO recorded with 1000-ms depolarizations from a holding potential of –80 mV to potentials between 0 and +60 mV (protocol in inset, 0.1 Hz). B, mean ± S.E.M. IK,PO-voltage relationship (n = 20). C, voltage dependence of steady-state inactivation with 1000-ms prepulses to various potentials and 750-ms test pulses to +60 mV. D, mean ± S.E.M. data for voltage dependence of IK,PO activation and inactivation (n = 10 each). E, mean ± S.E.M. time to peak IK,PO and inactivation time constants (n = 10 each). F, example of IK,PO recovery from inactivation. G, mean ± S.E.M. current during the second pulse (IP2) normalized to current during the first pulse (IP1), as a function of P1-P2 interval (protocol in inset) with biexponential fit to mean ± S.E.M. data (n = 9). H, IK,PO during the 10th pulse (IP10) to +60 mV normalized to that during first pulse (IP1) plotted over different frequencies with and without extracellular sodium. Act., activation; inact., inactivation; τf, fast time constant; τs, slow time constant; TP, test potential.

Pharmacological Characterization. After having characterized the current as a K+-dependent current with slow inactivation and recovery from inactivation, we set out to obtain the pharmacological profile of IK,PO. The K+ channel blocker 4-aminopyridine (4-AP) was applied at concentrations between 0.1 μM and 100 mM. The left panel of Fig. 3A illustrates 4-AP effects on IK,PO. Reversible suppression was seen, with an IC50 on peak to steady-state current of 0.81 ± 0.16 mM (n = 7; Fig. 3A, right, closed circles). Washout returned current amplitude to 81 ± 5% of control. 4-AP at lower concentrations significantly accelerated current inactivation: inactivation τs and τf averaged was 135 ± 70 and 35 ± 14 ms, respectively, after application of 100 μM 4-AP, compared with 315 ± 51 and 115 ± 7 ms, respectively, under control conditions (P < 0.05 for each), a behavior suggestive of open channel block (Fedida, 1997). To consider the reduction of charge carried by IK,PO, we determined the 4-AP IC50 based on the area under the current-time curve (Dukes et al., 1990; Gogelein et al., 2004). Integration of the area between the remaining current at maximal 4-AP concentration and the transient outward currents for determination of fractional block yielded an IC50 of 39 ± 15 μM (Fig. 3A, closed circles; P < 0.01).

Use-dependent 4-AP-unblocking is characteristic of Kv4.2 and Kv4.3 currents (Campbell et al., 1993; Tseng et al., 1996). Currents were recorded with 400-ms pulses to + 60 mV. After current stabilization, 2 mM 4-AP was added to the bath, and cells were repetitively depolarized for 10 pulses (1 Hz; n = 4). There was no significant difference between currents elicited with the first compared with the last pulse (19 ± 7% versus 12 ± 8% of first-pulse current; P = N.S.) incompatible with use-dependent 4-AP unblocking.

Determination of Cl– and K+ dependence. A and B, IK,PO recorded from the same cardiomyocyte before (A) and after (B) substitution of external chloride with equimolar cyclamate (protocol in inset). C, mean ± S.E.M. data (n = 5). D, IK,PO-recording obtained at +60 mV with regular K+-containing internal solution. E, IK,PO-recording with substitution of internal K+ with equimolar Cs+ from a different cell isolated from the same pig as in D on the same day. F, mean ± S.E.M. IK,PO density at +60 mV for KCl (n = 10) and for CsCl (n = 9) (P < 0.001). TP, test potential.

Effect of IK,PO Inhibition on Atrial Action Potentials. To investigate the potential physiological role of IK,PO in porcine atrial repolarization, we recorded effects on APs (Fig. 5A). The addition of 0.1 mM 4-aminopyridine prolonged terminal AP repolarization (Fig. 5B).

Quantitative Real-Time RT-PCR. Results of quantitative real-time RT-PCR on RNA extracted from isolated cardiomyocytes of animals that were also used for patch-clamp experiments demonstrated predominant expression of Kv1.5 subunit mRNA (Fig. 6, A and B). Kv1.5 mRNA expression was ∼15-fold that of Kv4.3 and KChIP2 (which were similar) and ∼153-fold that of Kv1.4 (n = 6; P < 0.001 for each). Kv4.2 mRNA was barely detectable.

Discussion

Major Findings. This study provides evidence for the presence of a time-dependent K+ conductance in pig atrium with physiological properties and pharmacological responses compatible with the participation of Kv1.5 α-subunits, and a role in porcine atrial repolarization. These findings identify IK,PO as the likely target of Kv1.5 blockers in previous in vivo studies of novel antiarrhythmic compounds and suggest that pigs may represent a model for the study of atrial-selective antiarrhythmic drugs that act by inhibiting Kv1.5-based currents.

Previous Studies on Porcine Electrophysiology. Pigs have been used for a variety of experimental studies of cardiac arrhythmias (Janse et al., 1998; Wirth et al., 2003), but information about the cardiac cellular electrophysiology of the pig is limited. Porcine sinoatrial cells exhibit IKs (Ono et al., 2000) and ventricular myocytes exhibit an ICl,Ca that contributes to repolarization (Li et al., 2003). Another study by the latter investigators documented the presence of ICl,Ca, IKur, IKr, and IKs in pig atrial myocytes (Li et al., 2004). The IKur (which the authors called IKur.p) reported in the latter study was relatively small and apparent primarily at slow frequencies (0.05 Hz) at room temperature. IKur.p showed weak inward rectification, use dependence, 4-AP sensitivity (IC50 of 72 ± 4 μM), and TEA resistance. This work differs from ours in the use of low EGTA concentration in the pipette and recording at room temperature.

Our study adds to previous results in showing the presence of a substantial time-dependent outward K+ current that is sensitive to blockers of Kv1.5, but not other possible underlying subunits, and that contributes to porcine atrial repolarization. Several lines of additional evidence presented here (including biophysical properties as well as mRNA expression) are consistent with a potential role for underlying Kv1.5 K+ channel subunits.

Relation of Biophysical Properties to IKur and IK,slow. Mouse ventricular myocytes express a current termed IK,slow with kinetic properties consistent with Kv1.5 α-subunits (Zhou et al., 1998). In other species, Kv1.5 underlies the atrially expressed ultrarapid delayed rectifier current (IKur), which is often described as noninactivating. Although Kv1.5 current has generally been described as a delayed rectifier, it can show substantial time-dependent inactivation, with complete inactivation for depolarizations of sufficient duration (Feng et al., 1998; Lin et al., 2001; Snyders et al., 1993). The inactivation kinetics that we found for IK,PO were faster than those published for hKv1.5 in heterologous systems (e.g., τf = 250 ms; τs = 1500 ms; Lin et al., 2001), although the latter studies were performed at room temperature, which in itself substantially slows inactivation (Snyders et al., 1993). It is also possible that IK,PO involves a contribution of β-subunits, which are known to interact with Kv1.5 and accelerate its inactivation (Uebele et al., 1998). A full study of the molecular biology of IK,PO would be very interesting, but it is beyond the scope of the present article.

Pharmacological Profile of IK,PO. Kv1.5-based currents are sensitive to 4-AP. For example, mouse ventricular IK,slow is inhibited by 4-AP with an IC50 of 32 ± 5 μM (Zhou et al., 1998). Significant interspecies differences in 4-AP sensitivity of Ito currents attributed to Kv1.5 exist, with IC50 values ranging up to 600 μM in rat atrium (Zhou et al., 1998). However, despite differences in affinity, all Kv1.5-carried currents are 4-AP sensitive, as was IK,PO in this study. Relevant IK,PO charge carriage inhibition (based on assessment of area under the current-time curve accounting for open channel block) occurred with an IC50 of 39 ± 15 μM. IC50 for heterologously expressed hKv1.5 peak currents ranges between 50 and 290 μM (Grissmer et al., 1994; Bouchard and Fedida, 1995), consistent with the results for IK,PO in the present study. Although 4-AP is nonspecific in that it blocks both Ito,f and Ito,s, the underlying mechanisms are distinct. Block of Ito,s occurs predominantly in the open state (Campbell et al., 1993). In contrast, 4-AP block of Ito,f occurs through closed state binding and displays use-dependent unblocking and reverse use dependence (Patel and Campbell, 2005). No use-dependent unblocking was observed in the present study, and block was consistent with open-state dependence. Both Kv1.4 and Kv1.5 currents show predominant open-state 4-AP block, but Kv1.4 is insensitive to flecanide (Akar et al., 2004) and sensitive to H2O2, inconsistent with the response of IK,PO. A contribution of other Kv1 subunits to IK,PO was excluded by the absence of any effect of hongatoxin application. In contrast, IK,PO was sensitive to perhexiline, AVE0118, flecainide, and 4-AP at concentrations fully compatible with Kv1.5 inhibition (Rampe et al., 1995; Zhou et al., 1998; Gogelein et al., 2004). A relevant contribution of Kv3.1 subunits that have been shown to underlie IKur in dogs is excluded, because this current is exquisitely sensitive to TEA (IC50 of 0.3 mM).

Potential Importance of Our Findings. Our findings provide detailed information about the pharmacological and biophysical characteristics of a porcine outward potassium current that is a candidate to mediate the reported effects of Kv1.5-inhibiting atrial antiarrhythmic drugs (Wirth et al., 2003).

Class III antiarrhythmic agents that delay atrial repolarization are effective in treating AF. However, previously developed class III antiarrhythmic agents have prolonged atrial repolarization by blocking IKr. The untoward side effects of IKr-inhibiting drugs include potentially lethal ventricular proarrhythmia (Hohnloser and Singh, 1995). The differential expression pattern of cardiac ion channel subunits (like Kv1.5) in atria versus ventricles provides a potential basis for treatment options for atrial arrhythmias that have reduced proarrhythmic risks (Nattel et al., 1999). The development of Kv1.5-based drugs as atrial antiarrhythmic agents has been limited by a lack of animal models with Kv1.5-regulated atrial repolarization. Porcine models have been used for in vivo testing of Kv1.5-blocking drugs and have demonstrated potent efficacy against atrial arrhythmias without significant ventricular actions (Wirth et al., 2003). IK,PO, as characterized in this study, contributes to atrial repolarization and has properties suggesting that it is carried by Kv1.5 α-subunits. This result provides for the first time a biophysical basis supporting the use of pigs as a model to test novel Kv1.5-inhibiting atrial-selective anti-AF agents.

Limitations of This Study. Variability in cell isolation can affect the results obtained. To minimize errors introduced by this process, we studied APs and mRNA levels from cells in pigs that were used for current recordings on the same day. Furthermore, native cells express a variety of ionic currents, and their electrophysiological isolation requires selective protocols and pharmacological agents with imperfect specificity. One-second pulses were chosen to allow for almost complete inactivation of the current. In some instances, incomplete inactivation might have caused biophysical inaccuracy, but longer pulses were poorly tolerated, and the results had minimal effect on the analyses. Another limitation of this study is the lack of protein expression data. We tried to obtain Western blots from porcine atrial protein preparations, but we were unable to obtain specific bands with commercially available antibodies, none of which have been raised against porcine-specific epitopes.

Footnotes

J.R.E. was supported by Nachlass Martha Schmelz, Dr. Paul and Cilli Weill-Stiftung, and Deutsche Forschungsgemeinschaft (EH201/2-1). The contributions of P.C. and S.N. were supported by grants from Natural Sciences and Engineering Research Council, Canadian Institutes of Health Research, and the Quebec Heart and Stroke Foundation.

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